OVERALL ABSTRACT Epigenetics is a critical determinant of aging and longevity, and senescence is a key driver of age-associated pathologies. Our program is leading efforts to understand the epigenetics of cell senescence and aging. Our overall hypothesis is that the epigenome is inherently dynamic/plastic to provide for flexible regulation, and during aging, this dynamic epigenome undergoes a loss of overall integrity. This loss of epigenome integrity, in turn, contributes to a secondary cascade of cell and tissue signaling events that also exacerbate aging, for example Senescence-Associated Secretory Phenotype (SASP) in senescent cells, a cause of ?inflamm-aging?. We have coined the term ?chromostasis? for the process whereby cells and tissues attempt to manage their dynamic/plastic epigenome to maintain epigenome integrity, transcriptional fidelity and, hence, promote healthy aging and longevity. The current P01 grant period was highly successful, as measured by our publications (56 total), collaborative efforts (20 collaborative publications), and our contributions to the fields of aging and epigenetics (104 publications since initial funding in 2008). Our specific major accomplishments in the current grant period are: (1) We uncovered dramatic changes to the epigenomic landscape within senescent human cells, some of which also occur in aged and diseased tissues. We discovered mechanisms underlying this altered epigenomic landscape, including disruption heterochromatin and the first example of a nuclear substrate of autophagy. (2) We discovered new mechanisms for activation of the SASP in senescent cells, and pioneered new small molecule/drug-based approaches to inhibit the SASP and promote healthy aging, including MLL and HDAC inhibitors. (3) We discovered a DNA methylation clock in mouse, and showed its slowing by diverse prolongevity interventions. (4) We dissected the structure and function of the HUCA (HIRA/UBN1/CABIN1/ASF1a) histone chaperone complex, a key mediator of histone dynamics in senescent cells, and defined the molecular basis of HUCA?s histone H3.3 variant selectivity. (5) We found age-correlated alterations in conserved chromatin factors that lead to inappropriate cryptic transcription from gene bodies, and showed this to be a novel cause of aging. In the renewal of this PO1, we will (1) leverage multi-disciplinary discovery platforms to uncover mechanisms underlying deficient chromostasis, (2) determine relevance of altered chromostasis in mouse/human aging, and (3) dissect mechanisms underlying the secondary pro-aging signaling events and identify pharmacological approaches to block these processes to promote healthy aging.